Background & objectives: In Odisha State, the control of malaria vectors has become dependent on synthetic pyrethroids, which are used for treatment of all approved long-lasting insecticidal nets (LLINs). The vast use of just one class of insecticide has led to the problem of resistance to insecticides in malaria vectors. One of the major malaria vectors in Odisha State is Anopheles culicifacies Giles. The aim of this study was to determine the resistance status of An. culicifacies to deltamethrin, a synthetic pyrethroid and other common insecticides used by the National Vector Borne Diseases Control Programme (NVBDCP) for indoor residual spraying in Odisha State. Methods: Mosquitoes were collected during April 2014 - June 2014 from 15 randomly selected villages in five p0 lasmodium falciparum endemic southern districts of Odisha State. The blood-fed wild caught females were exposed to the diagnostic dosage of DDT (4.0%), malathion (5.0%) and deltamethrin (0.05%) for one hour. Mortality was recorded at 24 h after the exposure. Results: Results indicated that An. culicifacies was resistant to all the three insecticides used in the malaria control programme in the five districts of Odisha State. Interpretation & conclusions: Resistance management strategy by appropriate rotation of different groups of insecticides including carbamates and incorporating a synergist with synthetic pyrethroids for treating mosquito nets should be considered for the control of malaria vectors in the area, especially where An. culicifacies is predominant. Periodical monitoring of susceptibility/resistance status of An. culicifacies to different insecticides is warranted.

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Background & objectives: Due to ever growing insecticide resistance in mosquitoes to commonly used insecticides in many parts of the globe, there is always a need for introduction of new insecticides for the control of resistant vector mosquitoes. In this study, larvicidal and adulticidal efficacies of three neonicotinoids (imidacloprid, thiacloprid and thiamethoxam) were tested against resistant and susceptible populations of Anopheles stephensi Liston 1901, Aedes (Stegomyia) aegypti Linnaeus, and Culex quinquefasciatus Say (Diptera: Culicidae). Methods: Laboratory-reared mosquito species were used. Insecticide susceptibility tests were done using standard WHO procedures and using diagnostic dosages of insecticide test papers and larvicides. Adulticidal efficacy of candidate insecticides was assessed using topical application method and larval bioassays were conducted using standard WHO procedure. Results: The results of topical application on 3-5 day old female mosquitoes indicated that resistant strain of An. stephensi registered lower LC ₅₀ values than the susceptible strain. Among the three insecticides tested, thiacloprid was found more effective than the other two insecticides. Culex quinquefasciatus registered lowest LC ₅₀ for imidacloprid than the other two mosquito species tested. In larval bioassays, the LC ₅₀ values registered for imidacloprid were in the order of Cx. quinquefasciatus < An. stephensi (SS) < An. stephensi (RR) < Ae. aegypti. In case of thiacloprid, the order of efficacy (LC ₅₀ ) was Cx. quinquefasciatus < An. stephensi (SS) < An. stephensi (RR), whereas in case of thiamethoxam, the larvicidal efficacy was in the order of An. stephensi (RR) < An. stephensi (SS) < Cx. quinquefasciatus. Interpretation & conclusions: The present study indicated that insecticide resistant strains of mosquito species tested showed more susceptibility to the three neonicotinoids tested, and the possibility of using neonicotinoids for the control of resistant mosquitoes should be explored.

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Background & objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. Interpretations & conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region.

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Sudden deaths in children due to acute encephalitis syndrome (AES) from a tribal dominated district of Malkangiri in Odisha, India, was reported during September-November, 2012. The investigation was carried out to search for the possible viral aetiology that caused this outbreak. Clinico-epidemiological survey and seromolecular investigation were carried out to confirm the viral aetiology. Two hundred seventy two suspected cases with 24 deaths were observed. The patients presented with low to moderate grade fever (87%), headache (43%), vomiting (27%), cold (18%), cough (17%), body ache (15%), joint pain (15%), rash (15%), abdomen pain (9%), lethargy (5%), altered sensorium (8%), convulsion (2%), diarrhoea (3%), and haematemesis (3%). Laboratory investigation showed Japanese encephalitis virus (JEV) IgM in 13.8 per cent (13/94) in blood samples and JEV RNA in one of two cerebrospinal fluid (CSF) samples. Paddy fields close to the houses, high pig to cattle ratio, high density (33 per man hour density) of Culex vishnui mosquitoes, low socio-economic status and low health awareness in the tribal population were observed. This report confirmed the outbreak of JEV infection in Odisha after two decades.

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Background & objectives: The quality of breeding sites is reflected through the pupal productivity and the life history traits of Aedes mosquitoes. Using nutrient reserves and pupal productivity of Aedes as indicators, the larval habitats including sewage drains were characterized to highlight the habitat expansion and vector management. Methods: The pupae and adults collected from the containers and sewage drains were characterized in terms of biomass and nutrient reserves and the data were subjected to three way factorial ANOVA. Discriminant function analyses were performed to highlight the differences among the habitats for sustenance of Aedes mosquitoes. Results: Survey of larval habitats from the study area revealed significant differences (P<0.05) in the pupal productivity of Aedes among the habitats and months. Despite sewage drains being comparatively less utilized for breeding, the pupae were of higher biomass with corresponding adults having longer wings in contrast to other habitats. The nutrient reserve of the adults emerging from pupae of sewage drains was significantly higher (P<0.05), compared to other habitats, as reflected through the discriminant function analysis. Interpretation & conclusions: The present results showed that for both Ae. aegypti and Ae. albopictus, sewage drains were equally congenial habitat as were plastic, porcelain and earthen habitats. Availability of Aedes immature in sewage drains poses increased risk of dengue, and thus vector control programme should consider inclusion of sewage drains as breeding habitat of dengue vector mosquitoes.

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Background & objectives: Phlebotomus argentipes (Diptera: Psychodidae), the established vector for kala-azar is presently being controlled by indoor residual spray of DDT in kala-azar endemic areas in India. Search for non-hazardous and non-toxic biodegradable active molecules from botanicals may provide cost-effective and eco-friendly alternatives to synthetic insecticides. The present study was aimed at evaluating various plant extracts from endemic and non-endemic areas of Bihar for their insecticidal activity against sandfly to identify the most effective plant extract. Methods: Bio-assay test was conducted with larvae and adult of P. argentipes with different plant extracts collected in distilled water, hexane, ethyl acetate, acetone and methanol. Thin layer chromatography (TLC), column chromatography and high performance liquid chromatography (HPLC) were conducted for detection of active molecules. Results: Adults and larvae of sandflies exposed to the aqueous extract of Nicotiana tabacum resulted in 100 per cent mortality. The hexane extract of Clerodendrum infortunatum was found to kill 77 per cent adults but was ineffective against larvae. Bio-assay test of the ninth fraction (hexane extract-methanol phase) separated by column chromatography was found to be 63 per cent effective. The purple spot on the TLC of this fraction indicated the presence of a diterpenoid. HPLC of this fraction detected nine compounds with two peaks covering 20.44 and 56.52 per cent areas with retention time of 2.439 and 5.182 min, respectively supporting the TLC results. Interpretation & conclusions: The column separated 9 ^[th] fraction of C. infortunatum extract was found to be effective in killing 63 per cent of adult P. argentipes. Compounds of this fraction need to be evaluated further for identification and characterization of the active molecule by conducting individual bio-assay tests followed by further fractionation and HPLC. Once the structure of the active molecule is identified and validated, it may be synthesized and formulated as a product.

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Background & objectives: Malaria is a major public health problem in Tripura and focal disease outbreaks are of frequent occurrence. The s0 tate is co-endemic for both Plasmodium falciparum and P. vivax and transmission is perennial and persistent. The present study was aimed to review data on disease distribution to prioritize high-risk districts, and to study seasonal prevalence of disease vectors and their bionomical characteristics to help formulate vector species-specific interventions for malaria control. Methods: Data on malaria morbidity in the State were reviewed retrospectively (2008-2012) for understanding disease distribution and transmission dynamics. Cross-sectional mass blood surveys were conducted in malaria endemic villages of South Tripura district to ascertain the prevalence of malaria and proportions of parasite species. Mosquito collections were made in human dwellings of malaria endemic villages aiming at vector incrimination and to study relative abundance, resting and feeding preferences, and their present susceptibility status to DDT. Results: The study showed that malaria was widely prevalent and P. falciparum was the predominant infection (>90%), the remaining were P. vivax cases. The disease distribution, however, was uneven with large concentration of cases in districts of South Tripura and Dhalai coinciding with vast forest cover and tribal populations. Both Anopheles minimus s.s. and An. baimaii were recorded to be prevalent and observed to be highly anthropophagic and susceptible to DDT. Of these, An. minimus was incriminated (sporozoite infection rate 4.92%), and its bionomical characteristics revealed this species to be largely indoor resting and endophagic. Interpretation & conclusions: For effective control of malaria in the s0 tate, it is recommended that diseases surveillance should be robust, and vector control interventions including DDT spray coverage, mass distribution of insecticide-treated nets/ long-lasting insecticidal nets should be intensified prioritizing population groups most at risk to avert impending disease outbreaks and spread of drug-resistant malaria.

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Background & objectives: The epidemiology of dengue fever (DF) is complex in the Indian subcontinent as all the four serotypes are circulating. This study reports observations on dengue cases from a virus diagnostic laboratory of a north Indian tertiary care hospital catering to areas in and around Lucknow, Uttar Pradesh. Methods: Serum samples were obtained from suspected cases of dengue referred to the virus diagnostic laboratory during 2011 to 2013, and detailed history was taken on a pre-structured datasheet. All samples were tested for anti-dengue virus (DV) IgM antibodies and DV-non structural protein 1 antigen (NS1Ag) by ELISA. NS1Ag positive samples were tested further by conventional RT-PCR for DV-RNA detection and serotyping. Results: Of the 4019 suspected patients of dengue, 886 (22%) showed laboratory evidence of dengue virus infection. Of these, 19, 17 and 27 per cent were positive in 2011, 2012 and 2013, respectively. Children and adults were similarly affected by dengue in all the three years. Males were more commonly affected than females. The predominant DV serotype detected was DV-2, DV-1 and DV-3 in 2011, 2012 and 2013, respectively. DV-4 serotype was not detected. About half the cases positive for DV infection, showed symptoms of dengue with warning signs/ severe dengue. A distinct seasonality with increase in number of dengue cases in the post monsoon period was seen. Interpretation & conclusions: Change in circulating serotype of dengue virus; a distinct adult dengue involvement; and a remarkable number of cases presenting with severe dengue manifestations are the main findings of this study.

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Background & objectives: Dengue infection is endemic in several areas and the dengue virus is transmitted by Aedes mosquitoes. Thus, it becomes important to understand the breeding ecology of dengue vector and characterize the physicochemical parameters of its breeding habitat. The objective of this study was to analyze the physicochemical parameters of the breeding habitat of the dengue vector and to find out the nutrient composition of the habitat in and around Kolkata, West Bengal. In addition, a geographic information system (GIS) was used to map the disease prone areas for its effective management and prevention. Methods: Water samples were collected from various breeding habitats of Aedes mosquitoes of Kolkata and adjoining areas and were analysed for various physicochemical properties like acidity, alkalinity, hardness, electrical conductivity, total dissolved solids, concentration of chloride (Cl ^- ), sodium (Na ⁺ ), potassium (K ⁺ ), fluoride (F ^- ) in relation to larval prevalence. Results: Parameters like water pH, total dissolved solids, total hardness, electrical conductivity, concentration of chloride, sodium and potassium were seen to vary throughout the year. Certain parameters were found to be dependent on container type, like concentration of fluoride. Significant positive correlations were seen between per dip larval density and total dissolved solids (TDS) and electrical conductivity. Interpretation & conclusions: Water pH, electrical conductivity, total dissolved solids were seen to play a major role in the ovipositional preferences. Container type did not seem to affect TDS. Tyres had the highest TDS in most of the cases. Nutrient composition like sodium concentration was mostly found in the coconut shells, potassium concentration also showed the same. Thus, container type and various parameters and nutrients play a major role in determining where a gravid female mosquito will lay its eggs. It was observed that by altering various chemical and physical properties of breeding habitats it was possible to control the larvae survivability.

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Background & objectives: Rapid diagnostic tests (RDTs) have become an essential surveillance tool in the malaria control programme in India. The current study aimed to assess the performance of ParaHIT-f, a rapid test in diagnosis of Plasmodium falciparum infection through detecting its specific antigen, histidine rich protein 2 (PfHRP-2), in Odisha State, India. Methods: The study was undertaken in eight falciparum malaria endemic southern districts of Odisha State. Febrile patients included through active case detection, were diagnosed by Accredited Social Health Activists (ASHAs) for P. falciparum infection using the RDT, ParaHIT-f. The performance of ParaHIT-f was evaluated using microscopy as the gold standard. Results: A total of 1030 febrile patients were screened by both microscopy and the RDT for P. falciparum infection. The sensitivity of ParaHIT-f was 63.6% (95% CI: 56.0-70.6) and specificity was 98.9% (95% CI: 97.9-99.5), with positive and negative predictive values (PPV and NPV) of 92.6% (95% CI: 86.0-96.3) and 93.0% (95% CI: 91.0-94.5), respectively. When related to parasitaemia, the RDT sensitivity was 47.8% at the low parasitaemia of 4 to 40 parasites/µl of blood. Interpretation & conclusions: The results showed that the performance of the RDT, ParaHIT-f, was not as sensitive as microscopy in detecting true falciparum infections; a high specificity presented a low frequency of false-positive RDT results. t0 he sensitivity of ParaHIT-f was around 60 per cent. It is, therefore, essential to improve the efficiency (sensitivity) of the kit so that the true falciparum infections will not be missed especially in areas where P. falciparum has been the predominant species causing cerebral malaria.

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Background & objectives: Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. The first major JE outbreak occurred in 1978 and since 1981 several outbreaks had been reported in the Cuddalore district (erstwhile South Arcot), Tamil Nadu, India. Entomological monitoring was carried out during January 2010 - March 2013, to determine the seasonal abundance and transmission dynamics of the vectors of JE virus, with emphasis on the role of Culex tritaeniorhynchus and Cx. gelidus. Methods: Mosquito collections were carried out fortnightly during dusk hours in three villages viz. Soundara Solapuram, Pennadam, Erappavur of Cuddalore district. Mosquitoes were collected during dusk for a period of one hour in and around the cattle sheds using oral aspirator and torch light. The collected mosquitoes were later identified and pooled to detect JE virus (JEV) infection by enzyme linked immunosorbent assay (ELISA). Results: A total of 46,343 mosquitoes comprising of 25 species and six genera were collected. Species composition included viz, Cx. tritaeniorhynchus (46.26%), Cx. gelidus (43.12%) and other species (10.62%). A total of 17,678 specimens (403 pools) of Cx. gelidus and 14,358 specimens (309 pools) of Cx. tritaeniorhynchus were tested, of which 12 pools of Cx. gelidus and 14 pools of Cx. tritaeniorhynchus were positive for JE virus antigen. The climatic factors were negatively correlated with minimum infection rate (MIR) for both the species, except mean temperature (P<0.05) for Cx. gelidus. Interpretation & conclusions: High abundance of Cx. tritaeniorhynchus and Cx. gelidus was observed compared to other mosquito species in the study area. Detection of JEV antigen in the two species confirmed the maintenance of virus. Appropriate vector control measures need to be taken to reduce the vector abundance.

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Background & objectives: Albizia lebbeck Benth. (Leguminosae) has long been used in Indian traditional medicine. The current study was designed to test antimalarial activity of ethanolic bark extract of A. lebbeck (EBEAL). Methods: EBEAL was prepared by soxhlet extraction and subjected to phytochemical analysis. The extract was evaluated for its in vitro antimalarial activity against Plasmodium falciparum chloroquine (CQ) sensitive (MRC2) and CQ resistant (RKL9) strains. Cytotoxicity (CC ₅₀ ) of extract against HeLa cells was evaluated. Median lethal dose (LD ₅₀ ) was determined to assess safety of EBEAL in BALB/c mice. Schizonticidal (100-1000 mg/kg) and preventive (100-750 mg/kg) activities of EBEAL were evaluated against P. berghei. Curative activity (100-750 mg/kg) of extract was also evaluated. Results: Phytochemical screening revealed presence of alkaloids, flavonoids, phenols, saponins, terpenes and phytosterols. The extract exhibited IC ₅₀ of 8.2 µg/ml (MRC2) and 5.1 µg/ml (RKL9). CC ₅₀ of extract on HeLa cell line was calculated to be >1000 µg/ml. EBEAL showed selectivity indices (SI) of >121.9 and >196.07 against MRC2 and RKL9 strains of P. falciparum, respectively. LD ₅₀ of EBEAL was observed to be >5 g/kg. Dose-dependent chemosuppression was observed with significant ( p<0.001) schizonticidal activity at 1000 mg/kg with ED ₅₀ >100 mg/kg. Significant (P<0.001) curative and repository activities were exhibited by 750 mg/kg concentration of extract on D7. Interpretation & conclusions: The present investigation reports antiplasmodial efficacy of EBEAL in vitro against P. falciparum as evident by high SI values. ED ₅₀ of <100 mg/kg against P. berghei categorizes EBEAL as active antimalarial. Further studies need to be done to exploit its antiplasmodial activity further.

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Background & objectives: One of the major strategies being pursued for malaria control by the National Vector Borne Disease Control Programme is the distribution of long lasting insecticidal nets (LLINs) in endemic areas since 2009. Information on durability of insecticidal efficacy and physical integrity of LLINs and community usage at different time intervals of their use is essential to plan net replacements to maintain universal coverage for an effective and sustainable malaria control strategy. Therefore, a study was undertaken to assess these parameters in two malaria endemic districts of the Odisha State. Methods: A total of 309 households were selected in 15 villages of two community health centres (CHCs) (Borigumma and Laxmipur) from Koraput district and one (Khairput) from Malkangiri district. Data on net usage were collected during March to July 2014 using semi-structured questionnaires. PermaNet 2.0 were sampled from all households, replaced with new ones, and bioassays were carried out to determine the insecticidal efficacy of LLINs after four and half, four and two years of field use following the standard procedure of World Health Organization. Results: LLIN use rate varied from 57.9 to 90.2 per cent in the study CHCs. The annual washing rate per net in Borigumma, Khairput and Laxmipur was 6.6, 3.2 and 4.8, respectively. The LLINs used two years in the field caused 100 per cent mortality and four to four and half years caused below 80 per cent mortality, except one net. Interpretation & conclusions: Nearly 20 per cent of the people were out of net coverage and hence the Programme to ensure 100 per cent coverage. The community should adequately be educated so as to increase the net use rate and avoid incorrect washing practices.

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Background & objectives: Although having immense clinical relevance, yet only a few studies have been targeted to understand the chikungunya virus (CHIKV) susceptibility and growth in Aedes aegypti populations from India. This study was undertaken to investigate CHIKV susceptibility and growth kinetics in Ae. aegypti along with genetic heterogeneity of Ae. aegypti populations. Methods: Dose dependent CHIKV susceptibility and growth kinetic studies for three CHIKV strains reported from India were carried out in Ae. aegypti mosquito populations. The phenotypic variation and genetic heterogeneity in five Ae. aegypti populations were investigated using multivariate morphometrics and allozyme variation studies. Results: The dissemination and growth kinetics studies of the three CHIKV strains showed no selective advantage for a particular strain of CHIKV in Ae. aegypti. At 100 per cent infection rate, five geographic Ae. aegypti populations showed differences in dissemination to three CHIKV strains. Morphometric studies revealed phenotypic variation in all the studied populations. The allelic frequencies, F statistics, and Nei's genetic identity values showed that genetic differences between the populations were small, but significant. Interpretation & conclusions: The results obtained in this study suggest that genetic background of the vector strongly influences the CHIKV susceptibility in Ae. aegypti.

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Background & objectives: Studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting/enhancing replication of important flaviviruses. Hence, a study was designed to determine whether Bagaza virus (BAGV), a flavivirus isolated from Culex tritaeniorhynchus mosquitoes in India, alters susceptibility of Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes to Japanese encephalitis (JEV) and West Nile viruses (WNV). Methods: JEV and WNV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes in the presence of BAGV was carried out by intrathoracic (IT) inoculation and oral feeding methods. Mosquitoes were infected with BAGV and WNV/JEV either simultaneously or in a phased manner, in which mosquitoes were infected with BAGV by IT inoculation followed by super-infection with JEV/WNV after eight days post-infection (PI). JEV and WNV yield on 7 ^[th] and 14 ^[th] day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID ₅₀ ) method. Results: In Cx. tritaeniorhynchus mosquitoes, prior infection with BAGV significantly reduced JEV and WNV replication while in Cx. quinquefasciatus, BAGV influence was only seen with WNV. Reduction in virus titre was observed in IT inoculated and oral fed mosquitoes irrespective of the infection mode. JEV replication was also found reduced in Cx. tritaeniorhynchus mosquitoes persistently infected with BAGV at passage four. Interpretation & conclusions: BAGV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes altered their susceptibility to JEV and WNV producing low virus yield. However, the role of BAGV in inhibiting JEV/WNV replication in field mosquitoes needs further investigations.

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